When performing radioimmunoassays, enzyme-immunoassays and competitive protein binding assays, it is often necessary to separate the antigen-antibody complex or protein ligand complex from other components in the assay. Methods previously used to achieve this separation include the addition of a liquid phase reagent to cause a precipitate in the assay medium and the introduction of a heterogeneous phase material into the assay medium which is easily separated at the end of the assay.
Examples of the liquid phase method include (a) the fractional precipitation of proteins using salts or solvents which allow the proteins to retain their binding capabilities even in their precipitate forms and (b) the precipitation of the antibody with a second antibody against it.
Examples of the separation of reaction components employing the addition of heterogeneous phase materials include (a) the adsorption of free nitrogen with surface active sorbents such as charcoal, talc, microfine silica and resin; (b) the formation of antigen-antibody complexes on supports on which first or second antibody has been permanently linked; (c) the formation of antigen-antibody complexes on pre-precipitated second antibody particles; and (d) the formation of antigen-antibody complexes on the inside surface of tubes on which antibody has been immobilized.
With the exception of the coated tubes and coated solid supports, all liquid phase and heterogeneous phase reagents require centrifugation to separate precipitate or solid material from the assay medium. This step is not only time consuming, but often requires special or delicate handling of the material when removing supernatant fluid. If the assay result is time dependent, only a small number of tests can be processed to assure accurate timing. Although the coated tube avoids these separation problems and is simple to use, it suffers from very low binding capacity and poor consistency from lot to lot because of manufacturing difficulties.
Solid supports, sorbent or protein coated, that do not require centrifugation to separate them from the liquid medium have been most recently developed to perform in heterogeneous assays. However, they must be dispensed initially by the technician performing the assay. Normally this is not a serious limitation, but in the situation where the solid support has been coated with pathogenic antigens or radio-labeled reagents, it is best if the dispensing of the solid support can be obviated. Also, aspiration of the supernatant liquid is required to separate the solid support and absorbed components from the reaction medium at the end of the assay.